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M.S. / Applied Physics / A new method of measuring the level of 8-methoxypsoralen in blood serum was developed for the reasons of speed, accuracy, and cost. This new method uses laser induced fluorescence of the psoralen to determine the concentration in serum. The fluorescence is analyzed with an optical multichannel analyzer coupled to an intensified photodiode array detector. Research was first attempted on samples with ethanol as the solvent to confirm that the method would work. Sample concentrations of 8-methoxypsoralen in serum are determined by comparing the fluorescence signal obtained from previously known concentrations. Levels down to 200ng per milliliter of serum can be measured with this technique.

Nuisance odor levels produced by solid waste management operations are subject to regulatory standards due to their impacts on the quality of life of the residents living nearby the facility. Failure to meet regulatory standards may result in fines, litigation, inability to acquire permits, mitigation, and re-siting operations. Since measurement of environmental nuisance odors is currently limited to subjective techniques, monitoring odor levels to meet such standards is often problematic. This is becoming more acute as increasing residential populations begin to encroach on properties adjacent to landfills. In order to ensure that nuisance odor issues are minimized, it is necessary to provide an objective measurement. The objective of the current research is to develop a biosensor for providing an objective, standard measurement of odors. The approach is to modify the human odorant binding protein (hOBPIIa), isolated using published biomolecular techniques, by fluorescently tagging it with a chromophore functional group. When this protein is tagged with a fluorophore marker and excited in a spectrofluorometer, it emits light of a certain wavelength that can be detected and quantified. Once odorant molecules are exposed to this complex, they start replacing the fluorophore, and as a result, the emitted light intensity decreases in proportion to the number of odorant molecules. Since the protein response depends on odorant concentration, following an inverse Beer’s Law relationship, the odorants can be quantified accurately and rapidly using fluorometric measurements. The results establish quantitation ranges for different pure and mixture of odorant gases as well as the amount of gas that can be quantified across various flow rates. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

Waterborne disease is a significant contributor to the global burden of disease, in particular among high-risk populations in developing nations. State-of-the-art methods for the enumeration of microbial pathogens in drinking water sources have important limitations, including high initial cost, 24-48 hour delays in results, high staffing and facility requirements, and training requirements which all become especially problematic in the developing nation context. A number of alternative approaches to microbial water quality testing have been proposed, with the goal of decreasing the required testing time, decreasing overall costs, leveraging appropriate technology approaches, or improving sensitivity or specificity of the water quality testing method. One approach that may offer solutions to some of these limitations involves the deployment of sensor networks using fluorescent spectroscopy to detect intrinsic protein fluorescence in water samples as a proxy for microbial activity. In recent years, a number of researchers have found significant and meaningful correlations between indicator bacteria species and the protein fluorescence of drinking water samples. Additionally, advances in the semiconductor industry could be used to drive down the cost of such sensors. This technology may also be extensible to other water quality parameters, including dissolved organic matter or the presence of fluorescent pollutants. In this thesis, a literature review describes the fundamentals of fluorescence spectroscopy, historical and recent work regarding the fluorescence of the amino acid tryptophan and associated bacterial fluorescence, possible mechanisms for this association, and potential applications of this technology for drinking water quality monitoring and waste water process control. Extensibility of the technology is also discussed. Next, experimental methodology in reproduction of similar results is described. Samples were taken from seven (7) surface water sources and tested using membrane filtration and an off-the-shelf fluorescence spectrometer to help examine the association between the presence of indicator bacteria and the tryptophan fluorescence of the water sample. The results, showing an association of R2 = 0.560, are compared to the results of recent similar experiments. Finally, two prototypes are described, including their design requirements and data from prototype testing. The results of the testing are briefly discussed, and next steps are outlined with the goal of developing a low-cost, in-situ microbial water quality sensor using fluorescence spectroscopy principles.

Glutathione (GSH) is an important biological thiol, it performs significant biological functions such as serving an antioxidant which protect cells from oxidative stress by trapping free radicals which damage DNA and RNA. It is known that abnormal plasma levels of GSH have been linked to various human diseases. Therefore, the rapid, sensitive and highly selective detection of GSH is of great importance for investigating its functions in diseases diagnosis. Interestingly, we found in cetyl trimethylammonium bromide (CTAB) medium, the resorufin-based probe shows an extremely fast, highly selective response to GSH. The result indicates that this dye can be employed to detect GSH in biological samples such as human plasma. Cysteine (Cys) is another important biological thiol which is involved in a variety of significant cellur functions, including protein synthesis, detoxication, and metabolic process, etc. Abnormal levels of Cys are related to many diseases, such as slowed growth, Alzheimer's disease and cardiovascular disease. Thus, the detection and quantification of Cys in physiological media is of great importance. In this thesis, I am going to present two organic fluorescent probes (Resorufin-based probe and SNF probe) for the detection and quantification of Cys. In addition, we prove that they can directly quantify Cys in human plasma. The chemical mechanisms involved in the detection of Cys are discussed.

Bacterioferritin, an iron storage protein having a 24-subunit quaternary structure, was used as a model for the study of host-guest interactions and guest encapsulation, making use of its spherical cage-like structure. A hexahistidine-affinity tag fused to the C-terminus of each bacterioferritin subunit was constructed. The C-terminus of each subunit points toward the inside of the cavity, while the N-terminus is exposed on the surface of the protein. The hexaHistag was able to form strong interactions with a nickel-nitrilotriacetic acid linked dye molecule (guest) and this interaction was used in attempts to develop a principle to control guest molecule encapsulation within the spherical cavity of the 24-mer bacterioferritin protein molecule. The procedure involved (1) subunit dissociation under acidic pH, (2) affinity controlled dye-Histag binding with exposed C-terminal hexahistidine residues and (3) reassociation of the subunits at neutral pH. The encapsulation conditions involving step 1 and 3 were studied preliminarily using laser light scattering to measure size (hydrodynamic radius) of the protein particle with apoferritin as a model system as it resembles the size and structure of bacterioferritin. In order to encapsulate guest molecules, the emptied shell of bacterioferritin was generated by site-directed mutagenesis resulting in ferroxidase- as well as heme-free bacterioferritin mutants (E18A/M52L/E94A), and these mutants were used to examine protein stability before conducting encapsulation experiments. However, wild-type bacterioferritin possessed highest stability in maintaining its multisubunit structure; hence, it was used for the encapsulation studies. It was found that 100% bacterioferritin with hexahistidine tag at the C-terminus, and a combination of 60% bacterioferritin with hexahistidine tag at the C-terminus and 40% bacterioferritin without hexahistidine tag at the C-terminus yielded similar amounts of encapsulated guest molecules. This suggested that all hexahistidine at the C-terminus were not equally available for dye molecule binding.

Bacterioferritin, an iron storage protein having a 24-subunit quaternary structure, was used as a model for the study of host-guest interactions and guest encapsulation, making use of its spherical cage-like structure. A hexahistidine-affinity tag fused to the C-terminus of each bacterioferritin subunit was constructed. The C-terminus of each subunit points toward the inside of the cavity, while the N-terminus is exposed on the surface of the protein. The hexaHistag was able to form strong interactions with a nickel-nitrilotriacetic acid linked dye molecule (guest) and this interaction was used in attempts to develop a principle to control guest molecule encapsulation within the spherical cavity of the 24-mer bacterioferritin protein molecule. The procedure involved (1) subunit dissociation under acidic pH, (2) affinity controlled dye-Histag binding with exposed C-terminal hexahistidine residues and (3) reassociation of the subunits at neutral pH. The encapsulation conditions involving step 1 and 3 were studied preliminarily using laser light scattering to measure size (hydrodynamic radius) of the protein particle with apoferritin as a model system as it resembles the size and structure of bacterioferritin. In order to encapsulate guest molecules, the emptied shell of bacterioferritin was generated by site-directed mutagenesis resulting in ferroxidase- as well as heme-free bacterioferritin mutants (E18A/M52L/E94A), and these mutants were used to examine protein stability before conducting encapsulation experiments. However, wild-type bacterioferritin possessed highest stability in maintaining its multisubunit structure; hence, it was used for the encapsulation studies. It was found that 100% bacterioferritin with hexahistidine tag at the C-terminus, and a combination of 60% bacterioferritin with hexahistidine tag at the C-terminus and 40% bacterioferritin without hexahistidine tag at the C-terminus yielded similar amounts of encapsulated guest molecules. This suggested that all hexahistidine at the C-terminus were not equally available for dye molecule binding.